Sugar chain is a generic term including glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and molecules in which monosaccharides, which are derivatives thereof, are linked by glycosidic bonds. Sugar chains encompass a wide variety of substances and are involved in various functions of living organisms. Analysis of sugar chains by electrophoresis is widely known as a technique for studying functions or analyzing structures or the like of sugar chains (see, for example, Japanese National Phase PCT Laid-open Publication No. 5-500563). This method visualizes the migration patterns of the sugar chain for analysis.
A method of transferring sugar chains from an electrophoresis gel to a film using a semidry blot transfer device is also known (see, for example, Japanese National Phase PCT Laid-open Publication No. 5-503146). In this method, sugar chains separated by electrophoresis are further transferred to a film of negative charge derivatives etc. of PVDF (polyvinylidene difluoride) after fluorescence detection in order to analyze the sugar chains on the film. In this way, it is possible to examine sugar chains, which react with a lectin or an antibody only when these are bound to proteins or lipids, by reacting the sugar chains with a lectin or an antibody without being influenced by a protein or a lipid, by transferring the sugar chains to the film. It is also possible to liberate a sugar chain band from the film, and apply mass spectrometry to the sugar chains. However, in this method, transfer is performed by electrophoretic transfer. Thus, charged sugar chains readily pass to the film, and as a result, the amount of transferred sugar chains is small. Therefore, the method is not suitable for precise quantitative determination and analysis.
Sugar chains of complex carbohydrates which widely exist in nature are important components of living bodies, and it is becoming evident that these play an important role in interactions between cells. Thus, various techniques for analyzing a small amount of sugar chain structures have been developed. In these techniques, steps of liberating sugar chains, separating and purifying sugar chains, labeling sugar chains and the like are combined as appropriate. However, such steps require cumbersome processes. Particularly, a separation and purification process for the sugar chains included in a mixture in a small amount after liberation is difficult and requires a great deal of experience. Generally used methods include ion exchange resin, reverse-phase chromatography, active carbon, gel filtration chromatography, and the like. However, these separation methods are not methods for specifically recognizing a sugar. Thus, contamination with other components (such as peptides or proteins) is considerable. Further, the recovery rate varies depending upon the structures of the sugar chains. Finally, identification by NMR spectrum or MS spectrum is required in order to gain reliable information on the obtained sugar. Particularly, a method using MS spectrums is an effective method for sugar chain analysis because identification of a molecular weight is possible with a small amount in the order of nanograms to micrograms. Methods which can determine sugar chain structures conveniently without using MS are also proposed.
Methods for analyzing sugar chain structures of glycoproteins and the like include a method for analyzing sugar chain structures of an unknown sample by utilizing the principle that, when a sugar chain of a glycoprotein is liberated and then separated and analyzed using liquid chromatography, an elution behavior according to the sugar chain structure thereof is observed by comparing the elution behavior with the elution behavior of known sugar chains. Generally used methods include ion exchange resin, reverse-phase chromatography, active carbon, gel filtration chromatography, and the like. However, these separation methods are not methods for specifically recognizing a sugar. Thus, contamination by other components (such as peptides or proteins) is considerable. Further, recovery rates vary depending upon the structures of the sugar chains. Moreover, identification by NMR spectrums or MS spectrums is required in order to confirm the structure. Particularly, a method using MS spectrums is an effective method for sugar chain analysis because identification of a molecular weight is possible with a small amount in the order of nanograms to micrograms. Methods which can determine sugar chain structures conveniently without using MS are also proposed.
Methods in which high performance liquid chromatography (HPLC) is applied also exist. Such methods include methods by two-dimensional HPLC (see, for example, Anal. Biochem., 171, 73 (1988) Tomitani et al.) in which HPLC of two types of modes are combined, and a method in which a sample is treated beforehand with a mixed enzyme series such as exoglycosidase and the like, and the treated sample is analyzed by HPLC for determining the structure of the sugar chain (see, for example, Chemistry and Living Organism 32 (10) 661 (1994), Konishi et al.). In these methods, sugar chains can be detected by performing a sugar chain-selective chemical reaction, and fluorescein-labelling sugar chains. In both methods, the sugar chain structure is determined based on the holding period depicted in the HPLC chromatogram. On the other hand, an analysis method employing a column in which a lectin having sugar recognition capability is fixed (see, for example, Anal. Biochem., 164, 374 (1987) Harada et al.) is also reported.
However, an analysis method employing a method of HPLC has the following problems: (1) only one sample is analyzed at a time, thus many numbers of samples cannot be analyzed at the same time; (2) condition settings for HPLC are delicate and holding periods may deviate and are likely to be incorrect; (3) HPLC when combined with a post-column reactor or a pre-column reactor consume a large amount of reagent (4) when a lectin fixed column is used, adsorbing glycopeptide may be affected by a change in affinity of the lectin to the sugar; and (5) sugar chains have to be labeled by fluorescein, radio isotopes, or the like, thereby requiring time and effort.
Further, a method for analyzing by fixing sugar chains to a solid phase has been attempted. However, fixing sugar chains which have high hydrophilicity is technically difficult. Thus, methods for directly fixing sugar chains are proposed. Such methods include, for example, a method for fixing to an amino plate by an acid amide bond utilizing reducing terminals of the sugar chains (see, for example, O'Shannessy et al., Anal. Chemistry, 1990, 91, 1-8), highly polymerizing sugar chains in order to give hydrophobicity to sugar chains themselves and adsorbing to a plate (see, for example, Japanese Laid-Open Publication No. 62-212568), and a method in which the sugar chains are biotinylated and the sugar chains are fixed to a solid phase to which avidin is bound by utilizing strong affinity between the avidin and the biotin.
However, the method in which sugar chains are fixed to a solid phase such as plates, has the problem that fixing yield is lowered, operations become cumbersome, and a long time is required due to pre-treatment of the sugar chains and fixing using a condensation agent, and that non-specific adsorption tends to occur due to coexistence of contaminants. Recently, a method for analyzing sugar chains by an analysis method employing surface plasmon resonance was proposed. However, since expensive specific equipment is used, it is difficult to obtain wide-use.
There are some examples reported about a method in which sugar chains are oxidized using enzymes such as galactose oxidase, and then coupled to a solid substrate to which hydrazide groups are introduced, such as cellulose, gel, or the like. (see, for example, O'Shannessy et al., Anal. Chemistry, 1990, 91, 1-8). In this method, there is a process of oxidizing sugar chains, which requires additional effort. Further, if the sugar chains are not oxidized, cellulose or gel to which hydrazide groups are introduced does not react sufficiently. The reaction varies depending on the sugar chains. Sometimes, the reaction does not occur at all depending on a type of sugar chains.
A convenient method for organic synthesis of glycopeptide has been reported (see, for example, Stefano E. Cervigni, Pascal Dumy, Manfred Mutter Angew. Chem. Int. Ed. Engl., 1996, 35, 1230-1232). However, chemical reactions utilizing specificity for sugar chains, which are used in this method, are intended for obtaining a product of a novel sugar chain cointegrate. There is no reported example on a carrier material specific to sugar chains targeting separation, purification and analysis of unknown sugar chain samples. Moreover, the peptide used in this method can be attached to a solid support and the like, but the form of the reaction is adsorption, and no phase transition occurs. This means that the peptide attached in this example are not bound to a solid support. Thus, when the attached peptides are exposed to a large amount of excessive solvent, they are liberated. Therefore, it is substantially impossible to perform purification, separation, analysis and the like even when the peptide used in this method is used. It is also impossible to produce a device which requires strong bonds to a support, such as a sugar chain chip.
As described above, a technique which enables one to directly separate and analyze sugar chains, irrespective of the types thereof, still does not exist. Such a technique is very important and desirable in the post-genomics and post-proteomics era.
The objective of the present invention is to provide a substance which can specifically bind to sugar chains irrespective of the type of the sugar chains. Another objective of the present invention is to provide a method for separating, purifying, concentrating or analyzing sugar chains or sugar chain-containing substances, efficiently and/or in faithful accordance with the state in nature, and a system and apparatus used therefor. Yet another object of the present invention is to utilize sugar chain components which naturally exist in a sample in a form where the ratio of content reflected.